Head and neck cancer is one of the most prevalent cancers worldwide. Normal human oral cavity epithelial cells respond to the signaling molecule retinoic acid (RA), a vitamin A derivative, by reducing their proliferation rate. In oral cavity squamous cell carcinomas (SCC), the growth inhibitory response to RA is generally reduced or lost. Methylation of the promoter of the RA-inducible gene retinoic receptor beta 2 (RARBeta2) at CpG islands is one of the first events that occurs as normal oral cavity epithelial cells become malignantly transformed. However, the reason why RARBeta2 promoter methylation occurs and the mechanism driving this event are important questions that are not yet answered. The Polycomb group (PcG) proteins regulate the deposition and recognition of multiple post-translational histone modifications. My hypothesis is that epigenetic changes mediated by Polycomb group proteins take place at the RARBeta2 promoter as human oral cavity epithelial cells become tumorigenic. These PRC driven epigenetic changes then lead to the silencing of transcription of the RARBeta2 gene in the majority of human oral cavity squamous cell carcinomas. I propose that this transcriptional silencing results from the abnormal recruitment of DNA methyltransferases to the RARBeta2 RARE which occurs as a consequence of PRC protein overexpression or greater association of the PRC complex proteins with the RARBeta2 gene in human oral SCC. I propose the following experiments to test this hypothesis: Aim 1: To assess the chromatin states at the RARBeta2 promoter in a variety of normal and transformed human cultured oral cavity epithelial cells; and Aim 2: To determine the functional effects of modulating the levels of expression of various transcriptional regulatory and chromatin remodeling proteins that show altered expression in human head and neck squamous cell carcinoma (HNSCC). For Aim 1 I will use chromatin immunoprecipitation (ChIP) assays to measure the recruitment of different transcriptional regulatory and chromatin remodeling proteins to the RARE of the RARBeta2 gene promoter. I will also employ real time PCR to measure the RARBeta2 mRNA levels in the proposed cell lines. For Aim 2, I will use tetracycline (doxycycline) regulated vectors for the overexpression/RNAi knockdown experiments. This inducible expression system will enable me to measure the kinetics of chromatin states at the RARE of the RARBeta2. In addition, I will modulate pharmacologically the epigenetic changes that occur with the knock down experiments. These proposed experiments are important because they provide mechanistic information about epigenetic changes that occur during the process of human oral cavity carcinogenesis. Specifically, the experiments I propose will provide insights into the temporal sequence of epigenetic events that occur during oral epithelial cells transformation. Finally, the experiments outlined in my proposal will potentially lead to the development of new therapeutic regimens.